Forster distances between green fluorescent protein pairs
Forster distances between green fluorescent protein pairs
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DOI：
10.1006/abio.2000.4708
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发表时间：
2000-09-10
影响因子：
2.9
通讯作者：
Barisas, BG
中科院分区：
文献类型：
--
作者：
Patterson, GH;Piston, DW;Barisas, BG
材料和方法GFP的表达和纯化。如前所述（5）制备用于Histaged绿色荧光蛋白颜色变体的质粒，并转化到大肠杆菌菌株BL 21 pLysS中用于蛋白质表达。His标记的GFP变体蛋白在E.如前所述（5）。接种后，培养物孵育2小时，用0.1mM异丙基β-D-硫代半乳糖苷诱导，并生长5小时，然后通过离心收获。将来自超声处理的细胞的上清液通过Ni-NTA，洗涤柱，并用含咪唑的缓冲液洗脱GFP。通过考马斯亮蓝染色的十二烷基硫酸钠凝胶的扫描光密度测定法评估蛋白质纯度（95%）。使用Beer定律计算消光系数，并如前所述（5）在Hewlett-Packard 8453 UV-可见分光光度计（Waldbronn，德国）中测量7 M，pH 8.0蛋白质溶液的吸光度。在dac 1681 Fluorolog分光荧光计（Edison，NJ）上进行荧光激发和发射测量。如（5）所述进行量子产率测量，分别使用pH 8.0下的每种变体的相等488 nm光密度和EBFP和其它GFP的1-氨基蒽（量子产率0.61）或荧光素（量子产率0.85）参考标准。DsRed的吸收和荧光发射光谱由蛋白质研究人员提供（3）。
Materials and MethodsGFP expression and purification. Plasmids for Histagged green fluorescent protein color variants were prepared as previously described (5) and transformed into the Escherichia coli strain BL21 pLysS for protein expression. The His-tagged GFP variant proteins were grown in E. coli as previously described (5). After inoculation, cultures were incubated for 2 h, induced with 0.1 mM isopropyl beta;-D-thiogalactopyranoside, and grown for 5 h before harvesting by centrifugation. Supernatant from sonicated cells was passed over Ni–NTA, the column washed, and GFP eluted by an imidazole-containing buffer. Protein purity (95%) was assessed by scanning densitometry of sodium dodecyl sulfate gels stained with Coomassie brilliant blue.Spectroscopy. Extinction coefficients were calculated using Beer’s law and the absorbance of 7 M, pH 8.0 protein solutions measured as previously described (5) in a Hewlett–Packard 8453 UV–visible spectrophotometer (Waldbronn, Germany). Fluorescence excitation and emission measurements were performed on a SPEX 1681 Fluorolog spectrofluorometer (Edison, NJ). Quantum yield measurements were performed as described (5), using equal 488-nm optical densities of each variant at pH 8.0 and 1-aminoanthracene (quantum yield 0.61) or fluorescein (quantum yield 0.85) reference standards for EBFP and other GFPs, respectively. Absorption and fluorescence emission spectra of DsRed were supplied by the proteins’ investigators (3).